key: cord-0736629-2szbkeim authors: Xu, Danyang; Sampson Duncan, Joseph K.; Licursi, Maria; Gohda, Jin; Kawaguchi, Yasushi; Hirasawa, Kensuke title: Antiviral roles of interferon regulatory factor (IRF)-1, 3 and 7 against human coronavirus infection date: 2022-03-27 journal: bioRxiv DOI: 10.1101/2022.03.24.485591 sha: df969ff08204688dbbf398e47d809e78dbcf3647 doc_id: 736629 cord_uid: 2szbkeim Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). As many human coronaviruses are known to be sensitive to IFN, antiviral roles of IRFs are yet to be fully understood. TypeI or II IFN treatment protected MRC5 cells from infection of human coronavirus 229E, but not human coronavirus OC43. Infection of 229E or OC43 efficiently upregulated ISGs, indicating that antiviral transcription is not suppressed during their infection. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of the IRFs demonstrated that IRF1 and IRF3 have antiviral property against OC43 while only IRF3 and IRF7 are effective to restrict 229E infection. Our study demonstrates that IRF3 plays critical roles against infection of human coronavirus 229E and OC43, which may be an anti-human coronavirus therapeutic target. 1 transducer and activator of transcription (STAT) (16, 17) . Phosphorylated STAT proteins 2 directly bind to the promoter regions of IFN-stimulated genes (ISGs) to activate their 3 transcription along with other transcriptional regulators such as IRF1 and IRF9 (18, 19) . 4 These ISGs function as antiviral factors and immune regulators. Human coronaviruses are generally sensitive to antiviral actions of IFNs albeit with 6 some differences in their sensitivity. Both SARS-CoV and MERS are sensitive to IFN when 7 cells are treated at high concentrations (20-23). Between the two viruses, IFNs are more 8 effective in inhibiting the replication of MERS than SARS-CoV (23). Moreover, SARS-CoV-9 2 is much more sensitive to type I IFN than SARS-CoV (24, 25) . As for other human 10 coronaviruses, IFNs suppress OC43 infection in a cell type dependent manner (26), while 11 229E is sensitive to IFN treatment in vitro (27, 28) . These reports suggest that human 12 coronaviruses are generally sensitive to IFNs but each virus has different levels of sensitivity. In clinical settings, IFN- treatment significantly reduced the mortality of SARS- 19 Western blot analysis 20 Western blot analysis on cell lysates was conducted and evaluated using antibodies listed 21 above as previously described (47). 23 Quantitative RT-PCR (RT-qPCR) was performed in triplicate using the previously described 24 validation strategies (50). The primer sequences are shown in Supplementary Table S1 . Figure 3A ). The expression of IRF1 and IRF7 was increased at 3 and 4 days 5 after infection compared to uninfected controls. Similarly, phosphorylated IRF3 was 6 increased at the same time points. After OC43 infection, OC43 nucleoprotein was detected on 7 day 1, which peaked at 3 and 4 days after infection ( Figure 3B ). In these infected cells, IRF1 8 expression increased at day 2 and 3. We observed an upper shift in IRF1 bands, which may 9 be caused by posttranslational modifications of IRF1. IRF3 and IRF7 were also activated 10 from 2 to 5 days after OC43infection. To determine the effect of SARS-CoV-2 infection on IRFs, Calu-3 cells were infected 12 with SARS-CoV-2. We detected viral protein after 1 day, which peaked at 2 days after 13 infection ( Figure 3C ). In these cells, there was an increase in IRF1 and IRF7 expression and 14 IRF3 phosphorylation at 2 days after infection. Thus, SARS-CoV-2 infection also activates 15 antiviral IRFs. These results demonstrate that IRF1, IRF3 and IRF7 are activated during human 17 coronavirus infection. To investigate the functional roles of IRF1, IRF3 and IRF7 during human coronavirus 20 infection, we conducted a loss-of-function analysis using siRNA knockdown. MRC5 cells 21 were transfected with either control siRNA oligos or those against IRF1, 3 or 7 for 24 hours. 22 The knockdown of IRF1 and IRF3 was confirmed with western blot analysis showing lower 23 expression levels in cells treated with their corresponding siRNA oligoes ( Figure 4A ). As 24 IRF7 expression is undetectable by western blot in non-infected cells, IRF7 knockdown was 1 confirmed by qPCR analysis ( Figure 4B ). When these cells were challenged with 229E or 2 OC43, an RT-q-PCR analysis revealed that IRF1 knockdown promotes 229E infection at 3 3 days after infection and OC43 infection at 2 and 3 days after infection ( Figure 4C ). IRF3 4 knockdown also increased the expression of viral RNA at 2 and 3 days after 229E infection In this study, we demonstrated an antiviral role of the IFN-IRF axis against human 3 coronavirus infection. We first found that human coronavirus 229E is moderately sensitive to 4 type I and II IFN while OC43 is not (Figure 1 ). Infection of both viruses efficiently induced 5 ISGs and activated IRF1, IRF3 and IRF7, suggesting that the antiviral innate response of 6 infected cells is not fully suppressed during infection (Figure 2 and 3) . Activation of IRFs 7 was also observed during SARS-CoV-2 infection (Figure 3) . The loss-and gain-of function 8 experiments demonstrated that IRF1 and IRF3 have antiviral roles against OC43 infection 9 while IRF3 and IRF7 were effective to suppress 229E infection (Figure 4 and 5). We found that 229E is sensitive to type I and II IFN, in agreement with previous 11 studies (21,28). Type I IFN has been shown to inhibit OC43 infection in A549 (lung cancer In western blot analysis, we observed the IRF1 bands, which are higher than expected, 2 at 2 and 3 days after OC43 infection ( Figure 3B ). Interestingly, this shift was not observed in 3 cells infected with 229E or SARS-CoV-2 infected cells. We believe that the size increase of 4 IRF1 may be induced by its posttranslational modifications. The phosphorylation or 5 monoubiquitination promotes the transcriptional activities of IRF1, suggesting that the upper 6 bands of IRF1 could be the active form (66). While this may be the reason why IRF1 showed 7 antiviral activities against OC43 infection but not 229E infection ( Figure 5A ), it is yet to be 8 clarified why they were observed only in cells infected with OC43. We use MRC5 cells for most of our studies (Figure 1 -4) while H1299 cells were used 10 for the gain-of-function experiments for IRF3s ( Figure 5 ). This is because we encounter 11 technical problems to achieve sufficient expression levels of the IRFs by transfecting the 12 plasmids without causing cell morbidity or affecting virus infection in MRC5 cells. A) MRC5 cell were left 2 untreated or treated with IFN-α (500 and 250 U/ml), IFN-γ (100 and 50 U/ml) or IFN-λ (100 -STAT1 and GAPDH antibodies. (B and C) MRC5 cells were left untreated or 5 treated with IFN-α or IFN-γ Western blot analysis of viral protein was conducted using anti 229E N protein 7 (B), OC43 N protein (C) and GAPDH antibodies. (D and E) TCID 50 assay was performed to 8 measure the progeny virus of 229E or OC43 infected MRC5 cells left untreated or treated 9 with IFNs and then challenged with VSV at MOI of 0.0001. VSV infection was 11 determined by (F) western blot analysis using anti VSV G and GAPDH antibodies and (G) 12 plaque assay using L929 cells. The amount of progeny virus is shown as plaque forming units 13 (PFU)/ml of samples nontreated or treated with IFN Two-way ANOVA Human coronavirus infection activates transcription of ISGs. MRC5 cells 16 were left uninfected or infected with 229E (A and C) or OC43 (B and D) at MOI of 0 Infection of 229E (A) and OC43 (B) was confirmed by western blot analysis using anti 229E The relative quantification (RQ) indicates the fold change of the 21 expression level for infected samples towards that of the non-infected controls at the same 22 time point post infection. The transcriptional level for each gene was calculated by 23 normalizing to GAPDH expression level Two-way ANOVA MRC5 cells 2 were left uninfected or infected with 229E (A) or OC43 (B) at MOI of 0.01. (C) Calu-3 cells 3 were left uninfected or infected with SARS-CoV-2 at MOI of 0.001. The activation of IRF1 SARS-CoV-2 N protein, IRF1, IRF3, IRF7, phosphorylated pmol) using Lipofectamine RNAiMAX transfection reagent. The knockdown of IRFs 10 expression was confirmed by western blot analysis for IRF1 and IRF3 (A) and RT-qPCR for 11 IRF7 (B). MRC5 cells were then infected with 229E or OC43 at MOI of 0.01. (C) The 12 amounts of viral RNA were measured by RT-qPCR. (D) The amounts of viral protein were 13 determined by western blot analysis using antibodies against 229E N protein, OC43 N protein 14 and GAPDH. For RT-qPCR analysis, the transcription level for each gene was first 15 normalized to GAPDH expression level. The relative quantification (RQ) indicates the fold 16 change of the expression level for IRFs siRNA IRF3 (B) 21 or IRF7 (C) and then infected with or without 229E or OC43 at MOI of 0.01. Virus infection 22 was determined by western blot analysis using antibodies against 229E N protein, OC43 N 23 protein, IRF1, IRF3, IRF7, phosphorylated IRF3 (p-IRF3) and GAPDH. 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